use of applied reproductive technologies (ftai, ftet) to ... supl_s183-s202.pdf · r.c. chebel....

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R.C.R.C.R.C.R.C.R.C. C C C C Chebhebhebhebhebel.el.el.el.el. 2011. 2011. 2011. 2011. 2011. Use of Applied Reproductive Technologies (FTAI, FTET) to Improve the Reproductive Efficiency inDairy Cattle. Acta Scientiae Veterinariae. 39(Suppl 1): s183 - s202.

Acta Scientiae Veter inar iae, 2011. 39(Suppl 1) : s183 - s202.Acta Scientiae Veter inar iae, 2011. 39(Suppl 1) : s183 - s202.Acta Scientiae Veter inar iae, 2011. 39(Suppl 1) : s183 - s202.Acta Scientiae Veter inar iae, 2011. 39(Suppl 1) : s183 - s202.Acta Scientiae Veter inar iae, 2011. 39(Suppl 1) : s183 - s202.

ISSN 1679-9216 (Online)

CORRESPONDENCE: R.C. Chebel [[email protected] – PHONE: +1 ( 612) 625-3130]. Department of Veterinary Population Medicine,University of Minnesota, 1365 Gortner Ave, ZIP CODE: 551098, Saint Paul, MN, USA.

UUUUUse of Ase of Ase of Ase of Ase of Applied Rpplied Rpplied Rpplied Rpplied Reprepreprepreproooooducducducducductivtivtivtivtive e e e e TTTTTechnoloechnoloechnoloechnoloechnologies (FTgies (FTgies (FTgies (FTgies (FTAI,AI,AI,AI,AI, FTET FTET FTET FTET FTET) t) t) t) t) to Impro Impro Impro Impro Improoooovvvvve thee thee thee thee theReproductive Efficiency in Dairy CattleReproductive Efficiency in Dairy CattleReproductive Efficiency in Dairy CattleReproductive Efficiency in Dairy CattleReproductive Efficiency in Dairy Cattle

Ricardo C. ChebelRicardo C. ChebelRicardo C. ChebelRicardo C. ChebelRicardo C. Chebel

ABSTRACT

Background: Reproductive inefficiency of dairy cattle, characterized by reduced estrous expression and detection rates,reduced pregnancy per artificial insemination (number of cows pregnant divided by number of cows inseminated), reducedpregnancy rates (number of cows pregnant divided by the number of cows eligible to become pregnant during a time interval),and increased pregnancy losses, has a large financial impact on dairy operations across the world. Although the most importantcomponent of reduced reproductive efficiency in dairy cattle is unquestionably poor management and diseases that resultfrom it, the genetic selection and the resulting increased milk yield have caused physiological changes in lactating dairy cowsthat also affect fertility. The most important of these changes is the increased feed intake and the consequent increasedmesenteric and liver blood flow to supply the nutrients necessary for milk yield. This causes significant decreases inconcentrations of progesterone and estradiol that affect estrous expression, follicular growth, oocyte quality, and embryodevelopment and survival. This review will discuss reproductive technologies used in large dairy herds to mitigate the effectsof these physiological changes on reproductive performance.Review: The use of ovulation/estrous synchronization protocols (OSP), pre and post-ovulation hormonal treatments, andembryo transfer (ET) in the reproductive management of lactating dairy cows was reviewed. Several OSP have been developedin the past 20 years. To achieve acceptable pregnancy per artificial insemination (P/AI) OSP should result in synchronizedrecruitment of a new follicular wave, growth of follicles under P4 concentration > 2 ng/mL, synchronized luteolysis, andsynchronized ovulation at the end of the protocol. When embryo recipient cows are submitted to OSP, these protocols mustaim to tightly synchronize luteolysis and ovulation at the end of the protocol. The use of ET in lactating dairy cows in the U.S.has been limited to herds of registered animals, to mitigate the negative effects of exposure to heat stress, to improve geneticsof expanding herds, and in a few herds to salvage repeat-breeders. Lactating dairy cows are sensitive to heat stress because ofthe high metabolic rate resulting from the increased feed intake necessary to supply nutrients for milk production. Severalstudies have demonstrated that lactating dairy cows exposed to heat stress that receive ET have improved reproductiveperformance compared with cows receiving AI. Finally, the use of hormonal treatments to increase P4 concentration duringearly diestrus was reviewed because several studies have demonstrated a strong association among P4 concentration andembryo development and pregnancy establishment. The effects of hormonal treatments during ovulation synchronizationprotocols, after AI or at ET on P4 concentration and reproductive outcomes are controversial and likely dependent onmanagement, milk yield, and diet of the lactating dairy cows.Conclusion: The use of reproductive technologies in lactating dairy cows, particularly AI, is extremely well disseminated andhas resulted in significant improvements in milk yield in the past 50 years. Recent developments in the understanding ofreproductive physiology of lactating dairy cows have resulted in ovulation synchronization protocols that optimize fertilityafter AI or ET.

Keywords: Lactating dairy cow, synchronization, artificial insemination, embryo transfer.

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I. INTRODUCTIONII. DISCUSSION2.1 Physiological Changes Associated with Reduced

Fertility2.2 Ovulation synchronization protocols2.2.1 Presynchronization.2.2.2 Resynchronization.2.2.3 Reducing the period of dominance of the ovulatory

follicle (5d-Cosynch).2.2.4 Low P4 concentration and fertility.2.3 Embryo transfer2.3.1 Mitigation of effects of heat stress on reproductive

efficiency.2.3.2 Reproductive performance of repeat-breeder cows.2.3.3 Effects of P4 concentration on embryo survival.2.4 Hormonal treatments to increase P4 concentration

during diestrus and pregnancy after AI or ET.III. CONCLUSIONS

I. INTRODUCTION

Fertility in lactating dairy cows has been de-creasing in the past 50 years. This decrease in fertilityhas been associated with a steady increase in milkyield, which is a consequence of genetic selectionfor milk yield and improvements in nutritionalmanagement. According to the National AnimalHealth Monitoring System [49], reproductive failureis the most important cause of involuntary culling.The costs of reproductive inefficiency to the cattleindustry are extremely important and have beenrecognized as such for decades. Senger et al. [71]

suggested that the dairy industry loses approximately$ 300 million per year because of poor estrousdetection rate and accuracy. The estimated averagevalue of a pregnancy is $ 275 [20] and that of anabortion is between $ 555 and $ 640 [20,80]. Thesevalues are dependent on lactation number, milk yield,days in milk (DIM), price of milk, and cost of repla-cement animals.

In technical terms, compromised repro-ductive inefficiency involves fertilization failure -observed from the day of AI to 5-6 d after AI, earlyembryonic loss - observed from 5-6 d after AI to 17-24 d after AI, late embryonic loss - observed from17-24 d after AI to 42 d after AI, and fetal loss -observed from 42 d after AI to term [64]. Reducedfertilization and increased early embryonic loss areusually observed as increased return to estrus afterAI. Increased late embryonic loss is observed as alteredinter-estrus interval or increased abortions if the firstpregnancy diagnosis takes place before 42 d afterAI. Increased fetal losses are observed as increasednumber of abortions after 42 d after AI (Figure 1).Ultimately, in large dairy herds, reproductive failureis observed as reduced pregnancy per AI (P/AI;number of pregnancies divided by the number ofcows inseminated), increased number of abortions,and decreased pregnancy rates (number of cowspregnant within a time period divided by the numberof cows eligible to become pregnant during the sameperiod).

Figure 1. Characterization of reproductive failure in cattle.

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Different studies have evaluated the ferti-lization of oocytes and quality of embryos fromlactating dairy cows, heifers, and non-lactating dairycows [10,12,17,21,67]. Although not all studiescompared production and quality of embryos bylactating dairy cows directly with that of heifers andnon-lactating dairy cows, it is estimated that non-superovulated lactating dairy cows have approxi-mately 76% of recovered structures fertilized, whereasnon-lactating cows and heifers have 78% and 100%,respectively, of recovered structures fertilized.Furthermore, approximately 66% of fertilizedstructures recovered from lactating dairy cows areclassified as excellent/good quality embryos butapproximately 74% and 72% of fertilized structuresrecovered from non-lactating dairy cows and heifers,respectively, are classified as excellent/good qualityembryos. Consequently, among all oocyte-embryorecovered from lactating dairy cows, 50% areclassified as excellent/good embryos, whereas 58%and 72% of all oocyte-embryo recovered from non-lactating cows and heifers, respectively, are classifiedas excellent/good embryos. In recent studiesconducted by our group in several herds across theU.S.A. we observed P/AI of lactating dairy cows tobe between 35 and 40% at 30 to 38 d after firstpostpartum AI [13,14,62]. Therefore, it is expectedthat 25% of all excellent/good quality embryos willbe lost between 6 and 35 d after first postpartum AIof lactating dairy cows, representing 1.78% ofembryonic losses per day.

Summarization of data from 15 differentstudies conducted in the U.S.A. that reported lateembryonic loss demonstrates that pregnancy lossesfrom 27-31 to 38-50 d after AI is approximately 13%with a range of 3 to 43%. This represents pregnancylosses of approximately 0.85% per day during thisperiod. Furthermore, incidence of late embryonic/fetallosses from approximately 40 to 120 d after AI hasbeen reported to range from 8.3 to 10.7%, whichrepresents daily losses of approximately 0.11% ofpregnancies diagnosed at 40 d after AI. On the otherhand, according to data from six publishedmanuscripts (total of 7,426 AI) P/AI at 38 d after firstAI in virgin heifers ranges from 55 to 70% and onlyapproximately 3% of heifers lose pregnancy from38 to 120 d of gestation, resulting in daily pregnancyloss of approximately 0.05%.

From these data it is obvious that the stagesof greatest risk for reproductive failure are fertilization,embryo development, maternal recognition ofpregnancy, and placentation. Furthermore, it is clearthat lactating dairy cows are less likely to conceiveand to carry out the pregnancy to term than virginheifers. Although the factors associated with reducedfertility in lactating dairy cows are multiple and multi-faceted, they all originate from the ability or lackthereof of lactating dairy cows to cope with thenutritional demands associated with the extremelyelevated milk yield. With the onset of colostrum/milkproduction lactating dairy cows face severe nutritionaldemands that are usually not fully met by feed intakeand result in negative energy balance, metabolicdiseases, immune suppression, and increase incidenceof diseases. In this manuscript we will discuss theeffects of increased milk yield on physiologicalalterations that affect reproductive efficiency, and wewill discuss reproductive technologies used to mitigatethe effects of these physiological alterations onreproductive performance.

II. DISCUSSION

2.1 Physiological Changes Associated with ReducedFertility

There are several hormones that are extremelyimportant to the reproductive function of ruminants[e.g. progesterone (P4), estradiol, GnRH, LH, FSH,and prostaglandin (PG) F2α]. In this section, we willbriefly discuss the importance of P4 and estradiol,their concentrations, and metabolism in lactating dairycows.

Estradiol is produced by antral ovarianfollicles. Under reduced concentrations of P4 (< 1ng/mL) estradiol is responsible for signs of estrus anda positive feed-back on the hypothalamus, whichstimulates secretion of GnRH that causes the pituitarygland to produce an ovulatory LH-peak. Furthermore,priming of the uterus with estradiol during theproestrus is expected to reduce the binding capacityof oxytocin to its endometrial receptors [43], reducingthe positive feed-back of oxytocin on endometrialproduction of PGF2α. For example, ovariectomizedcows treated with exogenous estradiol, mimickingconcentrations of estradiol during the proestrus, hadsmaller concentrations of PGF2α metabolite afteroxytocin challenge compared with oxytocin-challen-

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ged ovariectomized cows not treated with estradiol[43].

Several studies have found a link betweenmetestrus and early diestrus P4 concentrations andembryo development and elongation and thesubsequent establishment of pregnancy. Mann andLamming [42] demonstrated that cows that had thelargest embryos at 16 d after AI were also the cowsthat had the greatest P4 concentrations starting atapproximately d 5 after AI and that larger embryosproduced greater quantities of interferon-τ. Thisindicates that increased P4 concentrations during earlydiestrus should result in hastened development ofembryos and improved signaling from the embryofor maternal recognition of pregnancy [42]. Despitethe fact that mRNA for P4 receptors can be identifiedin nuclei of cells of early bovine embryos, in vitroexposure of cleaved embryos to elevated P4concentrations did not affect subsequent developmentto the blastocyst stage, nor recovery rates of 14 d oldin vitro produced (IVP) embryos 7 d after transfer[16]. On the other hand, supplementation with P4between d 3 and 7 of pregnancy did not alter themorphology of embryos recovered in the morula toblastocyst stage, but conceptus from heiferssupplemented with P4 were significantly larger at d13 and 16 after AI [9]. Similarly, when multipleembryos were transferred into superstimulatedrecipient heifers it was observed that embryostransferred into these heifers were significantly largerat d 13 than embryos transferred into non-superstimulated heifers, indicating a strongassociation between P4 concentration and embryodevelopment after the blastocyst stage [40]. Theestablishment of a uterine environment conducive toembryo growth and elongation appears to be P4dependent, because alterations in uterine geneexpression are induced by increased P4 concen-trations. For example, messenger RNA expressionfor transport and secretory proteins (e.g. lipoproteinlipase and connective tissue growth factor) presentin the bovine endometrium, thought to contribute touterine histotroph and thus conceptus elongation,were expressed earlier during diestrus and at higherlevels in cows with elevated P4 concentrations [26].

Studies have compared the concentrations ofestradiol and P4, the diameter of ovulatory follicles,and corpora lutea (CL) volume between lactating

dairy cows and non-lactating cows or heifers. Lopezet al. [41] demonstrated that high producing lactatingdairy cows (47 kg/day) had smaller peak concen-tration of estradiol during estrus (6.8 pg/mL) compa-red with low producers (32 kg/day - 8.6 pg/ml) andheifers (11.3 pg/mL), despite having larger ovulatoryfollicles (high producers = 18.6 mm, low producers= 17.4 mm, and heifers = 15 mm). Consequently,high producing dairy cows had shorter duration ofestrus (high producers = 7 h, low producers = 11.9 h,and heifers = 11.3 h) and had fewer standing eventsduring estrus (high producers = 6.5, low producers =9.8, and heifers = 16.8 mounts). Furthermore, Sartoriet al. [66] demonstrated that lactating dairy cows hadsmaller concentration of estradiol during estrus thannon-lactating dairy cows (7.9 vs. 11.3 pg/mL). Besidesaffecting estrus detection rates, this reduced estradiolconcentration is expected to result in prolongedinterval from luteolysis to ovulation (lactating cows= 5.2 ± 0.2 d, non-lactating cows = 4.6 ± 0.1 d) [66]because the estradiol threshold necessary to stimulatean LH surge would take longer to be reached inlactating dairy cows. Extended interval from luteolysisto ovulation compromises oocyte quality becausepre-ovulatory follicles are exposed to reducing P4concentrations and increasing LH pulsatility for longerperiods resulting in premature oocyte maturation.

Lactating dairy cows also have reduced P4concentrations compared with heifers starting as earlyas d 5 of the estrous cycle [66]. The reduced P4 con-centrations to which cows are exposed during me-testrus and diestrus may result in exposure of folliclesto increasing pulsatile release of LH, which causespremature oocyte maturation and reduced embryoquality [8,38]. Oocytes collected on d 8 of the estrouscycle from cows with P4 concentration declining from1.7 to 0.6 ng/mL from estrous cycle d 6 to 9 weremore likely to be in stage II of meiosis comparedwith oocytes from cows that had P4 concentrationincreasing from 1.4 to 3.1 ng/mL during the sameperiod [38]. Furthermore, cows exposed to P4 < 1ng/mL before ovulation are at higher risk for shortluteal phase, because the lack of P4 priming resultsin premature increase in estradiol receptors in theendometrium following ovulation and consequentlypremature expression of oxytocin receptors in theendometrium, which leads to premature secretion ofPGF2α and luteolysis [31,88]. Exposure of cows to

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reduced P4 concentration after AI may affect embryogrowth and consequently production of IFN-τ,compromising maternal recognition of pregnancy andpregnancy establishment as described previously.

It is not clear whether reduced production ofestradiol and P4 or increased metabolism of estradioland P4 or both are the cause for reduced estradioland P4 concentrations in lactating dairy cows, butthe latter is more likely. Studies conducted inWisconsin have demonstrated that the rate of meta-bolism of steroidal hormones in lactating dairy cowsis greater than that of non-lactating dairy cows [61].This seems to be directly correlated with the increasedfeed intake of lactating dairy cows and the consequenthyperthrophy and hyperplasia of the liver and organs

of the gastrointestinal tract. This results in increasedblood flow through the liver and greater metabolismof steroidal hormones. Sangsritavong et al. [61]demonstrated that unfed cows have reduced bloodflow through the liver compared with cows fed 7.8lb/d, 23.4 lb/d, and 33.4 lb/d (Figure 2), and that fasterdecreases in P4 and estradiol concentrations areobserved after feeding [61]. Similarly, cows receiving100 and 50% of NRC (2001) recommendations hadsignificantly reduced P4 concentrations comparedwith cows receiving 25% of NRC recommendationsor unfed cows (Figure 3) [85].

This is clear evidence that high yield lactatingdairy cows have reduced estradiol and P4 concen-trations as a consequence of increased feed intake,

Figure 2. Effect of feed intake on liver blood flow. Adapted from Sangsritavong et al. [27].

Figure 3. Effect of feed intake on progesterone concentration. Adapted from Vasconcelos et al. [28].

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which is a consequence of increased milk yield. Thisposes significant challenges to the reproductiveefficiency of these animals because of the importanceof estradiol and P4 for reproductive efficiency,explaining in part the significant decreases inreproductive efficiency observed in the past decades.

2.2 Ovulation synchronization protocolsFixed time AI (TAI) protocols were developed

in 1995 with the goal of synchronizing folliculargrowth, luteolysis, and ovulation. The first protocoldeveloped was the Ovsynch, which consists of oneinjection of GnRH on d 0, one injection of PGF2α ond 7, a second GnRH injection approximately 56 hafter the PGF2α injection and TAI at 12-16 h later[54]. The first GnRH injection synchronizes a newfollicular wave, whereas the PGF2α injectionsynchronizes luteolysis, and the last GnRH injectionsynchronizes ovulation. Subsequent studiesdemonstrated that the ideal time to initiate theOvsynch protocol is between d 5 and 9 of the estrouscycle, because at this stage of the estrous cycle morelactating dairy cows ovulate to the first GnRHinjection of the protocol [86]. Later, it wasdemonstrated that the ovulation to the first GnRHinjection of the Ovsynch protocol is critical forembryo quality [9] and P/AI [15] of lactating dairycows, because cows that do not ovulate to the firstGnRH injection have prolonged dominance periodof the ovulatory follicle [9] and ovulate aged oocytes[45]. Thus, presynchronization protocols weredeveloped in an attempt to maximize the number ofcows that start the timed AI protocol between d 5and 9 of the estrous cycle.

2.2.1 Presynchronization.The first presynchronization protocol

developed at the University of Florida was based ontwo injections of PGF2α given 14 d apart (Presynch)[47]. In this study, cows submitted to the Ovsynchprotocol 12 d after receiving the Presynch had P/AIapproximately 12 percentage points greater thanthose not presynchronized [47]. By giving 2 injectionsof PGF2α 14 d apart the percentage of cows thatdisplay estrus from 2 to 6 d after the second injectionis expected to be 65% [15], depending on com-pliance to the protocol and the percentage of anovularcows in the herd. Therefore, it is expected that bystarting the Ovsynch protocol 10 to 12 d after the last

PGF2α injection the majority of cows would bebetween d 4 and 10 of the estrous cycle.

In an attempt to simplify the Presynch-Ovsynch protocol by giving most injections on thesame of the week, Navanukraw et al. [50] comparedthe fertility of cows submitted to the Ovsynch protocolalone with the fertility of cows submitted to aPresynch-Ovsynch with the last PGF2á injectiongiven14 d before the start of the Ovsynch (14-14Presynch-Ovsynch). In this study, cows receiving thePresynch-Ovsynch (14-14) had greater P/AI thancows receiving the Ovsynch alone.

Galvão et al. [29] compared the fertility ofcows submitted to the 14-14 Presynch-Ovsynch tothat of cows submitted to a 14-11 Presynch-Ovsynch(interval between the last PGF2α injection of thePresynch and the start of the Ovsynch = 11 d). In thisstudy, cows receiving the 14-11 Presynch-Ovsynchhad P/AI 6 percentage points higher than cowsreceiving the 14-14 Presynch-Ovsynch [29]. Thisimprovement in fertility seems to result from theincreased percentage of cows that ovulated to the firstGnRH injection of the Ovsynch protocol whensubmitted to the 14-11 Presynch-Ovsynch comparedwith the 14-14 Presynch-Ovsynch [29].

More recently, presynchronization protocolsbased on GnRH and PGF2α injections have beendeveloped. Double-Ovsynch is the most know ofthese protocols as more peer-reviewed data exists[74]. As the name suggests, cows are submitted to a‘presynchronizing-Ovsynch’ and 7 d after its endcows are submitted to a ‘breeding-Ovsynch’. Thisprotocol has the following potential benefits:improved synchrony of the estrous cycle, anovularcows are more responsive to it than to the Presynch-Ovsynch, and more cows are likely to have growthof the ovulatory follicle under P4 concentrations > 2ng/ml. The studies published recently comparing theDouble-Ovsynch and the Presynch-Ovsynch,however, reported improvements in P/AI only inprimiparous cows submitted to Double-Ovsynch, butnot in multiparous cows [74]. It is unclear why onlyprimiparous cows benefited from the Double-Ovsynch, but one could speculate that becausegreater percentage of primiparous cows are expectedto be anovular early in lactation compared with multi-parous cows, the former would benefit the most fromthe additional GnRH injections given during the

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Double-Ovsynch. It is important to point out,however, that cows submitted to the Double-Ovsynchare less likely to be observed in estrus because of themultiple GnRH injections that they receive and thiswould affect estrous detection rates.

Therefore, the recommended protocol for firstpostpartum AI for herds with good estrous detectionrate is the Presynch-Ovsynch, with the intervalbetween the last PGF2α injection of the Presynch andthe start of the Ovsynch of 10 to 12 d.

2.2.2 Resynchronization.Most researchers agree that resynchronization

protocols to which cows diagnosed non-pregnant aresubmitted to have to be optimized. Resynchronizationprotocols used are dependent on herd size, estrousdetection rate, and diagnosis of pregnancy byultrasonography or manual palpation per rectum.Most published research on resynchronizationprotocols evaluated the effect of timing of initiationof the resynchronization protocol (usually theOvsynch protocol or an adaptation thereof) on P/AI.It is clear from these published manuscripts thatstarting the resynchronization protocols before d 28post-AI will result in reduced P/AI (Figure 4). This islikely because a large percentage of cows that startthe resynchronization protocol before d 28 post-AIwould be in proestrus, estrus, or metestrus at the startof the resynchronization protocol, affecting ovulationto the first GnRH injection, P4 concentration duringovulatory follicle growth, and synchrony of luteolysis.

Theoretically, if the length of the estrous cycleof lactating dairy cows is 23 d [66], the ideal intervalfrom AI to start the resynchronization protocol wouldbe between 28 and 32 d post-AI (d 5 to 9 of the newestrous cycle). It is interesting to note, however, thatrecent studies from our laboratory in collaborationwith other researchers demonstrated that starting theresynchronization protocol at different intervals afterd 27 post-AI does not affect P/AI [6]. In light of thefact that only 52% of cows non-pregnant to a previousAI are observed in estrus between 20 and 24 d post-AI, this finding is not surprising (Chebel personalcommunication, 2010). Several factors are likely toaffect the pattern of return to estrus: 1. approximately15% of cows submitted to ovulation synchronizationprotocols do not have the estrous cycle properlysynchronized and 10 to 15% of cows inseminatedbased on signs of estrus are not truly in estrus; 15%of postpartum cows are anovular cows that haveshorter luteal phase after first postpartum AI; and, 3.approximately 18% of inseminated cows areexpected to have early/late embryonic death. Thus,it is not surprising that we can poorly predict the stageof the estrous cycle that cows are in at the start ofresynchronization protocols, making necessarystrategies to presynchronize the estrous cycle of non-pregnant cows before the start of resynchronization.A limiting factor to controlling the estrous cycle ofinseminated cows before the start of resynchro-nization protocols is the fact that PGF2á cannot beused until cows are diagnosed non-pregnant.

Figure 4. Effects of interval between AI and initiation of the resynchronization protocol (Ovsynch or a variationthereof) on pregnancy per AI.

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Although the indicated label use of CIDRinserts is to improve return to estrus when used from14 to 21 d after initial AI, the use of CIDR insertsaccording to label recommendations has proven tobe inefficient, as the interval to re-insemination andproportion of cows re-inseminated prior to pregnancydiagnosis is not improved [15,29].

Recently, presynchronization protocols fornon-pregnant cows before the start of theresynchronization have been explored. In one study,non-pregnant cows were either resynchronized withthe Ovsynch protocol starting at 33 d post-AI or witha presynchronizing injection of PGF2α at 34 d post-AI and the Ovsynch protocol at 46 d post-AI [72].Cows presynchronized with PGF2α had greater P/AIthan cows resynchronized with the Ovsynch alone(35.2 vs. 25.6%) [72]. Similarly, cows resynchronizedwith the Double-Ovsynch (start of the‘presynchronization-Ovsynch’ 22 d post-AI, non-pregnancy diagnosis 29 d post-AI by ultrasonogra-phy, and start of the ‘breeding-Ovsynch’ 39 d post-AI) had greater P/AI than cows resynchronized withthe Ovsynch alone starting at 32 d post-AI (38.5 vs.30%) [32]. Although these experiments clearlydemonstrate that improvements in P/AI toresynchronized AI could be obtained from presyn-chronizing the resynchronization protocol, theseprotocols resulted in inter-AI interval 7 to 13 d longercompared with resynchronizing with the Ovsynchalone, which could offset the improvements in P/AI.

Our laboratory has recently conducted severalexperiments evaluating different resynchronizationprotocols. In the most recent study [24], cows at 31

± 3 d post-AI were selected to receive one of threeresynchronization protocols: Cosynch72 starting atnon-pregnancy diagnosis; CIDRsynch =Cosynch72+CIDR starting at non-pregnancydiagnosis; or G7G = GnRH injection at enrollmentand start the Ovsynch at non-pregnancy diagnosis.All cows were examined for pregnancy 7 d afterenrollment, at 38 ± 3 d post-AI. Throughout this study,cows observed in estrus were re-inseminated on thesame day. Among cows re-inseminated at fixed (afterthe completion of the resynchronization protocol),cows in the G7G and CIDRsynch treatments had thegreatest P/AI (Cosynch72 = 22.1%, G7G = 31.2%,CIDRsynch = 29.5%) [24]. When we evaluated thedata from all cows, including those re-inseminatedin estrus, the differences in overall P/AI after re-insemination were smaller (Cosynch72 = 28%, G7G= 32%, CIDRsynch = 31%) [46]. That was mainlybecause the presynchronizing GnRH injection givento G7G cows and the treatment with CIDR duringthe resynchronization protocol reduced the percentageof G7G cows and CIDR cows that were re-inseminated in estrus (Figure 5) [46]. Among cowssubmitted to the Cosynch72 treatment the P/AI ofthose re-inseminated in estrus was significantly betterthan that of cows re-inseminated at fixed time, whichincreased their overall P/AI (Figure 6) [46].

In a subsequent study, we evaluated theeffects of a presynchronizing GnRH injection givenat different intervals post-AI. In this study, cowsreceived a presynchronizing GnRH injection at 17or 24 d post-AI and started the Ovsynch 7 d later [7,46]. Thus, there were 4 treatments: EGGPG – d 17

Figure 5. Percentage of cows re-inseminated in estrus at different intervals in relation to the start of theresynchronization protocol (Day 0) and at fixed time. Adapted from Mendonça et al. [42].

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GnRH, d 24 GnRH, d 31 PGF2α (if diagnosed non-pregnant), d 33 GnRH, and d 34 TAI; EOVS – sameas EGGPG without the presynchronizing GnRH ond 17 post-AI; LGGPG – d 24 GnRH, d 31 GnRH (ifdiagnosed non-pregnant), d 38 PGF2α, d 40 GnRH,and d 41 TAI; and, LOVS – same as LGGPG withoutthe presynchronizing GnRH on d 24 post-AI [7,23].Cows were re-inseminated at any time if observed inestrus. Percentage of cows re-inseminated in estruswas smallest for EGGPG treatment and greatest forLOVS treatment (EGGPG=23.7, EOVS = 41.6,LGGPG = 49.0, LOVS = 57.6%) and the interval tore-insemination was slightly shorter for EGGPG andEOVS cows (EGGPG = 13.7 ± 0.2, EOVS = 11.6 ±0.2, LGGPG = 15.4 ± 0.3, LOVS = 14.6 ± 0.3 d)[7,23]. Overall P/AI [including cows re-inseminatedin estrus or at fixed time (TAI) upon completion ofthe resynchronization protocol] was not differentamong treatments (EGGPG = 26.2, EOV = 29.1,LGGPG = 30.5, LOV = 30.5%) [7,23]. Regardlessof treatment or farm, cows re-inseminated in estrushad greater P/AI at 66 d post-AI than cows thatreceived TAI (36.0 vs. 23.9%). Among cows recei-ving TAI upon completion of the resynchronizationprotocol treatment did not affect P/AI (EGGPG = 26.1,EOV = 19.4, LGGPG = 25.3, LOV = 23.8%) [7,23].

The use of CIDR within the resynchronizationprotocol has also been evaluated by our group incollaboration with other researchers [6]. In this studynon-pregnant cows were initiated in theresynchronization protocol at 32 or 39 d post-AI andreceive or did not receive a CIDR insert during the

resynchronization protocol [6]. In this study, weobserved that the interaction between time of initiationof the resynchronization protocol and CIDR treatmentaffected P/AI, because CIDR treatment tended toincrease P/AI of cows starting the resynchronizationprotocol at 39 d post-AI (28 vs. 23.7%) but had noeffect on P/AI of cows that started theresynchronization protocol at 32 d post-AI (no CIDR= 26.9 and CIDR = 24.2%) [6].

In summary, resynchronization protocolsshould start after 28 d post-AI. Up to date, no presyn-chronization protocols or additional hormonal treat-ments (e.g. CIDR during the resynchronization) havebeen able to increase P/AI of cows that start theresynchronization protocol between 28-34 d post-AI.On the other hand, cows that start the resynchro-nization protocol at 35-41 d post-AI should be pre-synchronized (e.g. PGF2α or GnRH or Ovsynch) ortreated with CIDR during the resynchronizationprotocol to increase P/AI to resynchronized TAI.Regardless of the resynchronization protocol chosen,herds that have good estrous detection accuracy andrate should not use 100% timed AI for re-inseminationof non-pregnant cows because more than likely P/AIof cows re-inseminated in estrus will be higher thanthat of cows re-inseminated upon completion ofresynchronization protocols.2.2.3 Reducing the period of dominance of the ovulatoryfollicle (5d-Cosynch).

Cows submitted to the Ovsynch protocol areexpected to have an interval from ovulatory follicleemergence to ovulation of 8.5 d approximately.

Figure 6. Pregnancy per AI according to resynchronization protocol and re-insemination procedure(AI in estrus or at fixed time (TAI)). Adapted from Mendonça et al. [42].

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According to Cerri et al. [10] reducing the intervalfrom emergence to ovulation in 2.3 d (from 8.1 to5.8 d) results in significant improvements in embryoquality. Thus, we have tested the hypothesis thatreducing the interval from the first GnRH injectionof the timed AI protocol to insemination wouldimprove fertility.

Because to achieve this reduction in intervalfrom the first GnRH injection to AI we would haveto treat cows with PGF2α 5 d after the GnRH injection,which could result in suboptimal luteolysis, in a pilotstudy, we compared the percentage of cows that hadluteolysis when PGF2α was given on d 7 (COS72;GnRH on d 0, PGF2α on d 7, and GnRH+TAI on d10), on d 5 (COS5d1; GnRH on d 0, PGF2α on d 5,and GnRH+TAI on d 8), or on d 5 and 6 (COS5d2;GnRH on d 0, PGF2a, on d 5 and 6, and GnRH+TAIon d 8) after the first GnRH injection [62]. Asexpected the percentage of cows that had luteolysiswas smallest for those receiving one injection ofPGF2α on d 5 after the GnRH (COS72 = 79.0,COS5d1 = 59.1, COS5d2 = 95.7%) [62].

In a subsequent study, 933 cows weresubmitted to the Presynch and 12 d later to either theCOS72 or the COS5d2 described previously [62].Cows receiving the COS5d2 had smaller ovulatoryfollicles (18.4 ± 0.3 and 16.8 ± 0.3 mm), reflectingthe shorter interval from follicle recruitment toovulation, and were more likely to have luteolysis(96.3%) compared with COS72 cows (91.5%) [62].Because greater luteolysis in COS5d2 cows couldconfound the effect of treatment on P/AI, weanalyzed P/AI including only cows that had luteolysisand observed that COS5d2 cows had higher P/AIthan COS72 cows at 38 (39.3 and 33.9%) and 66(36.7 and 32.5%) d after AI [62]. Thus, COS5d2treatment increased P/AI by reducing the dominanceperiod of the ovulatory follicle [62].2.2.4 Low P4 concentration and fertility.

The start of the timed AI protocols at 5 to 9 dof the estrous cycle is not only important to maximizethe percentage of cows that ovulate to the first GnRHinjection of the protocol and to assure that synchro-nized luteolysis will occur at the end of the protocol,but also to assure that ovulatory follicles grow underelevated P4 concentrations. As mentioned above,reduced concentrations of P4 before ovulation mayresult in increased exposure of follicles to pulsatilerelease of LH, which causes premature oocyte

maturation and reduced embryo quality [8,38]. Inrecent studies conducted by my laboratory [23,56]we evaluated whether the reduced P/AI observed inanovular cows and cows induced to ovulate folliclesof the first follicular wave was caused by the exposureof ovulatory follicles to reduced concentrations of P4.In these two studies we demonstrated that reduced P/AI of anovular cows and cows induced to ovulate thedominant follicle of the first follicular wave is aconsequence of compromised embryo quality becauseof exposure to P4 concentration < 2 ng/mL duringfollicle growth (Figure 7 and 8). Interestingly, therewas no effect of P4 concentration during growth ofthe ovulatory follicle on percentage of cows with shortluteal phase after AI [46], indicating that the benefitsof P4 concentration were likely associated with healthof oocyte.

2.3 Embryo transfer

Embryo transfer (ET) is not a new technologyas in 1891 Heape [36] reported the transfer of two 4-cell Angora embryos into inseminated Belgium rabbitsand the production of four Belgium and two Angorayoung from the same dam. Only six decades latersuccessful ET pregnancy [80] and birth of an ET calf[90] were reported. Since then, the growth ofcommercial application of ET in the cattle industryhas been significant and, in 2007, 823,160 embryoswere transferred [78].

According to the National Animal HealthMonitoring System [49], approximately 11.5% ofU.S.A. dairy herds have transferred at least one embryointo a lactating dairy cow or heifer in 2006.Interestingly, a similar percentage of operationstransferred embryos into only heifers or cows (8.9and 8.6, respectively) and slightly more operationstransferred fresh than frozen embryos (8.2 vs. 7.7%).Although ET is primarily seem as a technique toimprove the genetic composition of the herd, in recentyears, ET has been used in dairy operations in anattempt to improve reproductive performance oflactating dairy cows.

2.3.1 Mitigation of effects of heat stress on reproductiveefficiency.

High production dairy cows (daily milk yield> 35 kg) often consume 20 to 30 kg of dry matter perday. The increased dry matter intake (DMI) results insignificant increases in metabolic rates and heatproduction, such that the daily heat production by

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lactating dairy cows increased 30 MJ since the 50s[4]. This has prompted researchers to re-evaluatecharacterization of heat stress in lactating dairy cowsand to determine that increases in milk yield from 34kg/d to 46 kg/d result in decreased threshold tempe-rature of 9°F [5]. Consequently, heat stress affectshigh producing dairy cows more dramatically thanlower producing dairy cows and non-lactatinganimals [1,66].

Oocytes and embryos of lactating dairy cowsare damaged by heat stress. Several reports havedemonstrated that exposure to heat stress degeneratesthecal and granulosa cells, and reduces percentageof fertilized oocytes and percentage of recoveredstructures classified as excellent-good quality embryosin lactating dairy cows, and in vitro and in vivoembryonic development [28,34,44,47,50]. Embryosolder than 3 to 4 d of age produced in vivo from

Figure 7. Correlation among P4 concentration during the superstimulation protocol (P4) andpercentage of cows producing at least one transferable (solid line; percentage of cows producingat least one transferable embryo = 33.7 + (35.2 x P4) – (5.4 x P42); r2 (Adj.) = 0.96) or onefreezable (dashed line; percentage of cows producing at least one freezable embryo = 33.6 +(34.6 x P4) – (5.5 x P42); r2 (Adj.) = 1.0) embryo. Adapted from Rivera et al. [47].

Figure 8. Percentage of cows conceiving after first postpartum AI according to progesteroneconcentration during ovulatory follicle growth. Progesterone category: 0 = 0 to 0.99 ng/ml;1 = 1 to 1.99 ng/ml; 2 = 2 to 2.99 ng/ml; 3 = 3 to 3.99 ng/ml; 4 = 4 to 4.99 ng/ml; and, 5 >5 ng/ml. Pregnancy per AI (P/AI) = 24.2 + (6.9 x P4) – (0.76 x P42); r2 (Adj.) = 75.8%.Adapted from Denicol et al. [46].

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donors not exposed to heat stress or produced in vitro,however, are more resistant to heat stress becausethey are capable to produce heat-shock protein 70and because they have greater number of cells [69].

Several studies have demonstrated that,during heat stress, reproductive performance oflactating dairy cows receiving embryos is improvedcompared with lactating dairy cows receiving AI[28,30,51,72]. In a recent study conducted in Brazil[82] lactating dairy cows were assigned to receivefixed time AI or ET after having their ovulationsynchronized with one of two protocols. Althoughno differences in percentage of cows pregnant wereobserved between cows receiving AI or ET duringthe mild weather months, during the summer monthsa significant decrease in percentage of cows pregnantafter AI was observed, whereas season did not affectpercentage of cows pregnant at ET (Table 1).

In a study conducted in two commercial dairyfarms in TX, cows were randomly selected to receiveAI or IVP embryos, fresh or vitrified, during heatstress season [77]. In vitro produced embryos wereinseminated with sex-sorted semen. Because embryo

recipient cows only received embryos if they had aCL on the day of ET, whereas all cows assigned tothe AI treatment received AI, the researchers calculatedtwo reproductive outcomes: pregnancy rates(including all cows enrolled in each treatment,regardless if recipient cows received an embryo ornot), and P/AI or pregnancy per ET (P/ET; includingonly cows that received AI or ET) [77]. Cowsreceiving fresh IVP embryos had the bestreproductive performance (P/ET, pregnancy rates, andpercentage of cows producing a live calf) followedby cows receiving vitrified IVP embryos and cowsreceiving AI, respectively (Table 2). Furthermore, 80to 85% of offspring of cows receiving IVP embryoswere female calves (Table 2). Even though the costof a dose of semen was $ 20 and the cost of IVPembryos was $ 60, the significant improvements inreproductive performance during heat stress and theincreased number of live heifers produced fromtransfer of IVP embryos compared with cowsreceiving AI, transfer of IVP embryos resulted in anestimated return over investment of $ 22-42/lactatingcow/year [19].

Items Winter Spring Summer Fall P - value

Pregnancy rate 28 d, %

TAI 37.1a 32.9a 21.6b 27.0ab 0.08

ET 45.1 44.7 41.2 45.1 0.48

Pregnancy rate 60 d, %

TAI 33.7a 28.4ab 18.2b 25.8ab 0.02

ET 38.3 37.1 35.9 39.1 0.50

Items AI IVP fresh embryo IVP vitrified embryo P - value

Number of cows receiving AI or ET 219 134 188

Pregnancy per AI or ET1,% 22.9a,A 45.5b 30.9B < 0.01

Pregnancy rates, % 18.3a 42.1b 29.3c < 0.01

Cows delivering a life calf, % 14.6a 27.5b 17.1a < 0.01

Percentage of live calves of female sex2, % 50a,A 79.1b 72.5B < 0.01

Table 1. Reproductive performance of lactating dairy cows from two TX dairy herds exposed to heatstress and submitted to fixed time AI (TAI), in vitro produced (IVP) fresh and vitrified embryos.Adapted from Vasconcelos et al. [63].

a,b Within a row, means without a common superscript differed (P < 0.05).

Table 2. Reproductive performance of lactating dairy cows from two TX dairy herds exposed to heat stress and submitted to fixed time AI(TAI), in vitro produced (IVP) fresh and vitrified embryos. Adapted from Bilby et al. [64].

1Data regarding cows with properly synchronized estrous cycle. 2Deliveries referent to the services performed during the study.

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Unquestionably, improved in vivo and in vitroproduction of embryos has favored the use ET-basedreproductive strategies to improve reproductiveperformance of lactating dairy cows, particularlyduring seasons of heat stress. The more disseminateduse of IVP embryos for the reproductive managementof lactating dairy cows, however, depends on thecreation of cryopreservation procedures that resultin higher and more consistent P/ET.

2.3.2 Reproductive performance of repeat-breeder cows.Dairy cows are classified as repeat breeders

once they have received more than 3 AI and do notconceive. Considering that the average pregnancyrate of dairy herds in the U.S.A. is approximately16% and P/AI approximately 30%, it is not surprisingthat nearly 10 to 20% of lactating dairy cows in theU.S.A. may be considered as repeat-breeders. Thecauses of the reproductive failure of repeat-breedercows are not completely elucidated. Even though thedefinition of repeat-breeder cows is sub-fertility ofanimals that do not present anatomical or infectiousabnormalities at the time of diagnosis, it is likely thatdystocia, occurrence of postpartum diseases (e.g.mastitis, metritis, endometritis, displacement ofabomasum), and exposure to heat stress, among otherthings, are predisposing factors to this condition.Therefore, it is obvious that correction of predisposingfactors that cause repeat-breeders is the best solutionto this problem. Nonetheless, in situations in whichoocyte quality and/or uterine environment arecompromised, the use of ET to improve reproductiveperformance seems to be a good alternative. Recently,researchers have demonstrated in a large retrospectivestudy (n = 9,551) that pregnancy outcomes of repeat-breeder cows was significantly improved when theyreceived ET (41.7%) compared with repeat-breedercows that received AI (17.9%) [58].

2.3.3 Effects of P4 concentration on embryo survival.

Although the role of P4 on the growth ofembryos is well known, conflicting results have beenreported regarding the effects of P4 concentration onestablishment of pregnancy after ET. Although a fewstudies with Bos indicus influenced beef heifers andlactating dairy cows in tropical environment indicatedthat P4 concentration at the time of transfer affectedP/ET [17,28,39] studies with Bos taurus influencedbeef and lactating dairy cows demonstrated nocorrelation between P4 concentration at the time of

transfer and P/ET [40,44,60,64,84]. Similarly, P4concentrations 5 to 7 d after transfer did not affect P/ET in lactating dairy cows according to a few studies[30,81] but increased P/ET in beef and lactating dairycows according to others [39,51].

An important consideration that must be madewhen evaluating published data regarding the effectof P4 concentration at the time of ET or 7 d later onP/ET is whether the synchrony of the estrous cycleof the recipient cows was determined and controlledfor. In a recent study conducted by our group [39],we determined synchrony of the estrous cycle ofrecipient lactating Holstein cows based on P4concentrations at each injection of the OVP and atthe time of ET. Cows that did not have a properlysynchronized estrous cycle had greater P4concentration on the day of ET (3.9 ± 0.2 vs. 2.2 ±0.2 ng/ml), but had lower P4 concentration 7 d afterET (4.5 ± 0.3 vs. 5.6 ± 0.2 ng/ml). That was mainlybecause among cows with properly synchronizedestrous cycle only 3.6% had luteolysis from the dayof ET to 7 d later, whereas among cows that did nothave a properly synchronized estrous cycle 20% hadluteolysis from the day of ET to 7 d later [39].Consequently, P/ET 60 d after ET of cows that didnot have a properly synchronized estrous cycle waslower than P/ET of cows that had a properlysynchronized estrous cycle (17.1 vs. 27.2%). In thisstudy, when we excluded from the analysis cows thatdid not have the estrous cycle properly synchronizedand evaluated the effects of P4 concentration 7 d afterET on P/ET, we observed a significant correlationbetween them (Figure 9) [39].2.4 Hormonal treatments to increase P4 concentrationduring diestrus and pregnancy after AI or ET.

Because of the importance of P4 concen-tration on embryo development, attempts have beenmade to increase P4 concentrations during metestrusand diestrus of cows receiving AI or ET throughdifferent hormonal treatments during the OVP or afterpresumptive ovulation.

Several research groups have attempted toincrease P4 concentration during metestrus anddiestrus by treating cows with human chorionicgonadotropin (hCG) or GnRH at different intervalsafter presumptive ovulation. This is expected to cau-se ovulation of large/dominant follicles present in theovary at the time of treatment, formation of anaccessory CL, and increase P4 concentrations.

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Treatment with 1,500 to 3,300 IU of hCG at 5 to 7 dafter presumptive ovulation results in greater percen-tage of cows forming accessory CL and, in moststudies, significant increases in P4 concentrationsapproximately 7 d later [12,24,50,52]. The effects ofhCG given 5 to 7 d after AI or presumptive ovulationon pregnancy outcomes, however, are a little moreconflicting with some studies reporting higherpregnancy for hCG treated cows (51,53,60,65] and

other studies reporting no effect of hCG treatmenton pregnancy outcomes [41,51,78] (Figure 10).

The effects of GnRH treatment after presum-ptive ovulation on P4 concentration and pregnancyoutcomes also are conflicting. Lactating dairy cows[14,17,35] receiving GnRH 5 to 7 d after presumptiveovulation and lactating dairy cows receiving GnRH11 d after presumptive ovulation [89] had slightlyhigher P4 concentrations 5 to 7 d after treatment than

Figure 9. Correlation between P4 concentration 7 d after ET and pregnancy per ET (P/ET) 53 d after ET.Average (± SEM) P4 concentrations on 7 d after ET according to quartiles were: quartile 1 = 2.7 ± 0.2, quartile2 = 4.7 ± 0.1, quartile 3 = 5.9 ± 0.1, and quartile 4 = 8.4 ± 0.3 ng/ml. P/ET = 12.7 + (6.0 x P4); r2 (Adj.) =98.5%. Adapted from Kenyon et al. [74].

Figure 10. Effect of hCG treatment 5 to 7 d after presumptive ovulation on pregnancy outcomes after AI or ETin dairy heifers [Schmitt et al. (69)], beef cows [Nishigai et al. (51);Wallace et al. (89)], and dairy cows [Santoset al. (64); Hanlon et al. (69); Galvão et al. (30); Vasconcelos et al. (81)].

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non-treated cows. The increases in P4 concentrationafter GnRH treatment are smaller and less consistentcompared with those observed after hCG treatmentbecause hCG has a longer half-life than GnRH (30vs. 5 h) and, consequently, a more potent LH-likeactivity that not only causes new ovulations butextends the functional life of CL already present inthe ovaries [18]. Lactating dairy cows treated withGnRH had higher pregnancy outcomes according tosome [30,81] but not all studies [24,46]. In a meta-analysis conducted by Peters et al. [53], the effects ofGnRH treatment 11 to 14 d after presumptiveovulation on pregnancy outcomes was evaluatedbased on 16 studies and 8,535 inseminations.According to this meta-analysis, the interactionbetween study and treatment affected the pregnancyoutcomes, which deems this meta-analysis quiteinconclusive [86]. When the authors included in thelogistic regression important independent variablesthat could affect the pregnancy outcomes (e.g. OVPto which cows were submitted, cattle breed), however,

GnRH treatment did not affect pregnancy outcomes[86]. In recent studies we conducted in dairies in CA,MN, and TX, we demonstrated that GnRH treatmentat 17 or 31 d after presumptive ovulation did notreduce pregnancy losses from 31 to 66 d after AI[87], but cows treated with GnRH at 17 and 24 dafter AI had greater P4 concentration at 31 d after AIand reduced pregnancy losses from 31 to 66 d afterAI (7.5 vs. 11.9%) [88]. It is interesting to point outthat according to this later study [88] there was astrong correlation between P4 concentration at 31 dafter AI and incidence of pregnancy loss from 31 to66 d after AI (Figure 11).

Treatment of cows and heifers with equinechorionic gonadotropin (eCG) during OVP hasgarnered interest for its potential to increase P4concentration after ovulation and increase pregnancyper AI or ET. In general, the studies that evaluatedthe effects of eCG treatment on P4 concentration andpregnancy outcomes attempted to promote hastenedgrowth and ovulation of dominant follicles and,

Figure 11. Correlation between progesterone concentration 31 d after AI (P4) and pregnancy loss from 31to 66 d after AI. Pregnancy loss = 87.41 – (26.5 x P4) + (2.1 x P42); r2 (Adj.) = 99.7%. Adapted fromScanavez et al. [88].

consequently, increased P4 concentration after AI orET by treating animals with eCG close to the expectedtime of ovulation [32,36,47,56,90]. Although thesestudies do not promote consensus regarding theeffects of eCG treatment on pregnancy outcomes, itappears that anovular cows and cows with low body

condition score would benefit the most from eCGtreatment. On the other hand, treatment of embryorecipient lactating dairy cows with eCG around thetime of follicular wave emergence to promotesuperstimulation and higher P4 concentration at thetime of ET does not seem to improve P/ET (control =

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32.8 vs. eCG = 37.3%) [39]. According to this study,treatment of cows with 800 IU of eCG around thetime of follicular wave emergence resulted in reducedpercentage of cows with synchronous estrous cycle(61.0 vs. 71.7%) and reduced the percentage of cowsselected to receive ET (79.1 vs. 87.5%) [39]. It is notclear how eCG affects synchrony of the estrous cyclein lactating dairy cows, but the eCG’s long half-lifeis likely to be involved.

Important factors that may affect the effectsof hormonal treatments during the OVP and afterpresumptive ovulation on pregnancy outcomes arebody condition score, lactation status, environmentalconditions, and cattle breed.

III. CONCLUSIONS

The constant pressure for more efficient milkproduction demands that high producing dairy cowsbe used by dairy operations. Because of thedependency of milk yield on dry matter intake, it isunavoidable that lactating dairy cows will continueto demonstrate physiological changes (e.g. reducedsteroidal hormone concentration and increasesensitivity to heat stress) that predispose them tocompromised reproductive performance. The use offixed time AI protocols and ET in the reproductivemanagement of lactating dairy cows will continue tobe important, particularly in situations of heat stress.

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